Chemical Properties and Microbiological Profile of kunu zaki, A Non-Alcoholic Beverage Volume 4 - Issue 1

Braide W*¹, Ukagwu N², Lugbe PB³, Akien Ali AI³ and Adeleye SA¹

• ¹Department of Microbiology, Federal University of Technology, Nigeria
• ²Department of Science Laboratory Technology, Federal Polytechnic, Nigeria
• ³Department of Science Laboratory Technology, Rivers State Polytechnic, Nigeria

Received: April 11, 2018;   Published: April 25, 2018

Corresponding author: Braide W, Department of Microbiology, Federal University of Technology, PMB 1526, Owerri, Imo State, Nigeria

DOI: 10.26717/BJSTR.2018.04.001001

Abstract PDF

Kunu zaki is a popular indigenous cereal based non-alcoholic beverage widely consumed, especially in the Northern Nigeria. Composite samples randomly collected were screened for pH, titratable and microbiological characteristics using standard methods. Antibiotics susceptibility pattern of pathogenic bacteria was also determined using Kirby-Bauer disc diffusion method. The pH was slightly acidic to neutral (6.83-7.13), while titratable acidity was directly proportional to incubation time (mean value, 0.50%-1.53%). Total plate counts were high for bacteria (1.5 x 107Cfu/ml - 9.5 x 107Cfu/ml), coliform (0 - 7.0 x 106Cfu/ml) and fungi (6.0 x 106 - 1.0 x 106) and indicates gross contamination. Bacterial isolates include species of Bacillus, Escherichia coli, Lactobacillus, Staphylococcus, Salmonella and Shigella, whereas molds isolated belongs to the genera, Fusarium, Aspergillus, Penicillium, Rhizopus and Mucor. Saccharomyces species were the only yeast isolated. The health implications of the presence of Escherichia coli, Staphylococcus aureus, Salmonella species in foods and beverages had been widely reported.

Some species of Aspergillus, Fusarium, Rhizopus and Penicilium produces mycotoxins, a secondary metabolite associated with serious mycotoxicosis of animals and humans. Bacillus, Lactobacillus and Saccharomyces are involved in food deterioration and spoilage as they utilize the sugars present in the substrate to produce acids and other metabolites. The results of the antibiotic susceptibility test are worrisome as some of the organisms showed resistance to the tested antibiotics. For instance, Staphylococcus aureus and Salmonella sp were resistant to amoxicillin antibiotic whereas Escherichia coli, Bacillus cereus and Salmonella sp were resistant to ceftazidine antibiotic. Adequate sanitary measures should be employed during and after the production of Kunu zaki to reduce the microbial load.

Keywords: Kunu Zaki; Chemical and Microbial Profile; Antibiotic Assay

Kunu zaki is a popular nonalcoholic drink processed from cereal grains such as millet, maize, sorghum and rice [1-4]. It is highly nutritious and widely consumed in the Northern part of Nigeria for its thirst quenching properties. Kunu is traditionally prepared by women under unhygienic condition and packaged in inadequately sterilized containers which predispose it to contamination Aminat et al. [5]. During processing sweeteners, adjunct and thickeners are added which further contaminate the product. Kunu is not shelf stable because of local technology employed. Adeyemi and Umar [6] had reported the shelf life of 24h at ambient temperature. Kunu contains lactic acid bacteria such as Lactobacillus, Streptococcus and Leuconostoc species which could cause spoilage. Bacteria such Staphylococcus, Pseudomonas, Bacillus and fungi such as Penicillium, Aspergillus, Trichoderma and yeast have been isolated from processed kunu Osuntoki and Korie, [7]. The presence of these organisms in small number could render a beverage unsuitable for human consumption [8-10]. Kunu has a very high moisture content and total solid which may encourage the growth of strains of microorganisms to hazardous levels during storage at ambient temperature Olasupo et al. [11]. This study reports on the microbiological quality and chemical properties of kunu zaki stored at ambient temperature. The antibiogram of notable food borne bacteria was also determined.

Collection of Samples

Composite samples were randomly collected from three densely populated locations in Owerri, Imo State, Nigeria. Labeled samples in plastic bottles and cellophane bags were transported immediately in an ice chest to the laboratory for analysis. Figure 1 shows the procedure for preparation of kunu zaki.

Figure 1: Flow chart for the production of conventional KunuzakiAminat et al. [5].

Microbiological Analysis

Samples were analyzed microbiologically using standard methods [12-14]. Isolates were characterized according to the methods of Sharma [14]. Colony count was done using digital colony counter for bacteria and hand lens for fungi. Total colony was expressed as colony forming units'in milliliters (Cfu/ml) with reference to Harrigan and McCance [15]. Fungal colonies were identified colonially using characteristics such as pigmentation on the surface and reverse, sporulation, mycelia and spore arrangement microscopically [16-18]. Bacteria and yeast were identified colonially, microscopically and a few biochemical test [13,14,19].

Determination of pH and Titratable Acidity

Digital Uniscope pH meter was used to determine the level of acidity of the samples. The pH probe was inserted into a 100mls beaker containing 50mls of the samples and the values recorded when the reading was stable. Ten milliliters of the sample were titrated with 0.1N of sodium hydroxide using phenolphthalein as an indicator. Titration continues until a faint pink colour was obtained. Titration was done in triplicates and mean value obtained was used to calculate the percentage titratable acidity [14,20].

Antibiotic Susceptibility Test

Potential pathogenic bacteria isolated from the samples were screened for their antibiotic susceptibility using the Kirby-Bauer agar disc diffusion method CLSI, [21]. Suspension of inoculums prepared in 10mls peptone water was standardized to 0.5 McFarland turbidity equivalents. One milliliter of each standardized inoculums was seeded onto the surface of freshly prepared Mueller Hinton agar plates and allowed to stand for 30mins. Different concentrations of antibiotic discs (oxoid) were placed at equidistant and incubated at 370C for 24h. Zones of inhibition (in millimeter) were measured and recorded and compared with standards CLSI [21].

Table 1: pH values of the Kunu-zaki.

Table 2: Titratable Acidity of Kunu zakisamples (% of lactic acid).

Note: Total aerobic bacteria, coliform and fungal count is shown in Table 3. Total colony count expressed in Cfu/ml is high ranging from 1.5 x 10⁷- 9.5 x 10⁷; 0 - 7.0⁶ x 10⁶ and 1.0 x 10⁶ - 6.0 x 10⁶ for aerobic bacteria, colifom and fungi respectively.

Table 3: Microbial population of kunu zakisamples.

Note: The distribution and percentage of the isolates is shown in Tables 4-5. Bacillus (14.75%), Salmonella(9%) and Staphylococcus(8.25%) species were the predominant bacteria, where as Saccharomyces (8.96%) species predominate the fungi group.

Table 4: Percentage Distribution of Bacteria and Fungi isolates.

Table 6: Antibiotic Susceptibility Pattern of Bacteria isolated from Kunu Zaki.

Note: Zero, resistance (no zone of inhibition).

Table 5: Distribution of Bacteria and Fungi isolated from kunu zaki.

Table 1 shows the pH values of kunu zaki analyzed. The pH is slightly acid (6.83-7.13). Titratable acidity calculated in percentage is shown in (Table 2). Titratable acidity is directly proportional to incubation time in days. Total aerobic bacteria, coliform and fungal count is shown in (Table 3). Total colony count expressed in Cfu/ml is high ranging from 1.5 x 10⁷ - 9.5 x 10⁷; 0 - 7.0⁶ x 10⁶ and 1.0 x 10⁶ - 6.0 x 10⁶ for aerobic bacteria, colifom and fungi respectively. The distribution and percentage of the isolates is shown in (Tables 4 & 5). Bacillus (14.75%), Salmonella (9%) and Staphylococcus (8.25%) species were the predominant bacteria, where as Saccharomyces (8.96%) species predominate the fungi group. Table 6 shows the antibiogram of suspected pathogenic bacteria isolated from the kuni zaki. Ceftazidine was resisted by Escherichia coli, Bacillus cereus and Salmonella sp whereas Amoxicillin was resisted by Staphylococcus aureus and Salmonella sp. The tested bacteria show varying degree of susceptible to the other antibiotics.

The pH of sample ranged between 6.83-7.13, indicating slight acidity to neutrality. This condition may account for proliferation of spoilage and pathogenic organisms. Mean titratable acidity at ambient temperature is directly proportional to incubation or storage time. Increase in lactic acid indicates the fermentation of lactose sugar present in the substrate by Saccharomyces species and other lactic acid bacteria. The result showed significant difference in total acidity throughout the duration of storage at 0.05% confidence limit. Total aerobic counts of 1.5 x 107 - 9.5 x 107 Cfu/ml indicate high level of microbial contamination. Total coliform count ranged from 0 - 7.0 x 106 Cfu/ml, while total fungal counts is 1.0 x 106 - 6.0 x 106 (Table 3). The isolation of E. coli, Staphylococcus aureus, Shigella and Salmonella species is an indication that the beverage is contaminated with potentially pathogenic bacteria [8,22,23]. had reported that water and crude method of production and packaging under unhygienic conditions predisposes Kanu zaki to microbial contamination. Staphylococcus aureus cause food intoxication, where as Salmonella and Shigella species have been implicated in typhoid fever and shigellosis respectively Willey et al. [24].

Lactic acid bacteria such as Lactobacillus, Saccharomyces and Bacillus species thrives in sugar rich substrate and produce lactic acids Walker [25] via Embden-Meyerhof fermentation pathway Willey et al. [24], thus lowering the pH and increasing the titratable acidity (Tables 1 & 2). The presence of Escherichia coli in water and food is an indication of faecal contamination. Escherichia coli are an indicator organism and are an important member of the coliform group Makut et al. [10]. The report of [8,9] revealed that the presence of E. coli in hawked kunu zaki was as a result of contaminated water, containers as well as dirty environment where kunu was being processed and hawked. Oshoma et al. [26] had reported that E. coli may remain viable in acidic foods for days. Penicillium, Fusarium, Rhizopus and Aspergillus species isolated from the samples have been implicated with mycotoxins production and resultant mycotoxicosis in animals and humans [27,28].

Moulds are naturally found in the soil and may contaminate the raw materials from the field and during storage; and are common spoilage organisms of carbohydrate foods. Their spores can survive in low acid environment and spread by slight air current [9,10,29] reported that spices used as additives and flavour enhancers in foods and beverages are sources of contamination among other environmental factors. Oranusi et al. [30] and Braide et al. [31-33] had reported on the microbial contamination and antibiotic susceptibility of some locally fermented beverages and fruit juice consumed in Nigeria. Antibiogram of the bacterial test isolates showed varying level of susceptibility to the antibiotics tested against (Table 6). Complete resistance of Bacillus cereus and Escherichia coli to ceftazidine and Staphylococcus aureus and Salmonella sp to Amoxicillin may stern from drug abuse and genetic modification of the organisms to resist the antibiotics. This trend is worrisome as unwholesome and adulterated Kunu zaki contaminated with food borne pathogens may result into outbreak of food borne illnesses, especially in localities where the beverage is consumed voraciously.

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